This invention relates to a novel DNA sequence, and derivatives thereof, useful in the screening of compounds that are agonistic or antagonistic to seronergic receptor activity.
The enormous phenotypic diversity of neural cell types implies a corresponding complexity of gene-specific transcription factor combinations required to regulate thousands of genes in the appropriate stage and cell type-specific manner (He, X., and Rosenfeld, M. G., xe2x80x9cMechanisms of complex transcriptional regulation: implications for brain developmentxe2x80x9d Neuron 7:183-196, 1991; Mandel, G., and McKinnon, D, xe2x80x9cMolecular basis of neural-specific gene expressionxe2x80x9d Annu. Rev. Neurosci. 16:323-345, 1993; Struhl, K., xe2x80x9cMechanisms for diversity in gene expression patternsxe2x80x9d Neuron 7:177-181, 1991). Indeed, several members of different transcription factor classes, such as homeodomain, zinc-finger and basic helix-loop-helix proteins, function to regulate neural cell-type identity in specific regions of both the vertebrate and invertebrate nervous systems. Many of these genes are expressed early in neural development, which suggests they play a critical role in neurogenesis and neuronal patterning (Tanabe, Y., and Jessell, T. M., xe2x80x9cDiversity and pattern in the developing spinal cordxe2x80x9d Science 274:1115-1124, 1996). However, an understanding of the functional interplay between different transcription factors and the neural genes they regulate is just beginning to emerge. Little ia known about the identity and specific functions of transcription factors which operate particular differentiation programs involved in the appearance and maintenance of specific neural cell phenotypes.
The central serotonin (5-HT) neurotransmitter system consists of a relatively small population of morphologically diverse neurons whose cell bodies are present largely within the limits of the midbrain/hindbrain raphe nuclei and particular regions of the reticular formation (Steinbusch, H. W. M., Neuroscience 6:557-618, 1981). Although there are only about 20,000 serotonergic neurons in the rat brain the extensive axonal projection system arising from these neurons bears a tremendous number of collateral branches so that the 5-HT system densely innervates nearly all regions of the central nervous system (Halliday, G., Harding, A., and Paxinos, G. (1995) in The rat nervous system (Paxinos, G. ed), 2nd Ed., pp. 929-974, Academic Press, San Diego; Jacobs, B. L., and Azmitia, E. C., Physiological Reviews 72:165-220, 1992). Given its widespread distribution it is not surprising that 5-HT has been implicated in the control of numerous neural systems which mediate such functions as cognition, aggression, and perception (Heninger, G. R., Proc. Natl. Acad. Sci. USA 94:4823-4824, 1997). Abnormal function of the central 5-HT system has been implicated in several psychiatric maladies such as depression, anxiety, and eating disorders. The people afflicted by diseases caused or potentiated by abnormalities in the central serotonin 5-HT neurotransmitter system numbers in the millions. In this regard, this system is the target of several highly effective pharmacological agents that are used widely to treat these conditions. However, the current pharmacological agents may posses cross reactivity with other components of the central nervous system resulting in unwanted or debilitating side effects. Additionally, despite the clear importance of the central serotonin 5-HT system in a wide range of CNS processes and clinical disorders little is known about the genetic mechanisms which control the specification and differentiation of serotonergic neurons.
What is needed are reagents and methods that can be utilized in the screening of pharmacological compounds that can be utilized in the treatment of neurological diseases involving the central serotonin (5-HT) neurotransmitter system.
The present invention generally relates to compositions and methods of identifying and testing seronegic receptor agonists and antagonists. In addition, the invention relates to methods to identify other members of the EST transcription factor family, methods to identify homologs of Pet-1 which are native to other tissue or cell types or specific transcription factors for other neuronal cell types and methods to generate reagents derived from the invention.
The present invention contemplates employing a gene sequence (SEQ ID NO:1) that encodes a transcription factor specific for central serotonin 5-HT neurons. In one embodiment, the present invention contemplates a composition comprising isolated and purified DNA having an oligonucleotide sequence of: Pet-1 cDNA having the nucleotide sequence of SEQ ID NO:1. Such DNA may readily be inserted into expression constructs and the present invention contemplates such constructs as well as their use. The present invention also contemplates RNA transcribed from the above-indicated cDNA as well as protein (typically purified protein) translated from this RNA. Moreover, the present invention contemplates antibodies produced from immunizing with this translated protein.
The present invention also contemplates transgenic animals comprising the above-indicated DNA (i.e. the xe2x80x9ctransgenexe2x80x9d) or portions thereof. In a particular embodiment, the transgenic animal of the present invention may be generated with the transgene contained in an inducible, tissue specific promotor.
The present invention also contemplates using the above-named compositions in screening assays. The present invention is not limited by the particular method of screening. In one embodiment cells are used such as, but not limited to, transformed cell lines. In another embodiment primary cells may be used. The present invention is not limited to the nature of the transfection construct. The transfection constructs utilized will be the optimal constructs available for the cell line chosen at the time of setting up the assay. In one embodiment, the present invention contemplates screening suspected compounds in a system utilizing transfected cell lines. In one embodiment, the cells may be transfected transiently. In another embodiment, the cells may be stably transfected. In yet another embodiment translation products of the invention may be used in a cell-free assay system. In yet another embodiment, antibodies generated to the translation products of the invention may be used in immunoprecipitation assays.
The present invention may also be used to screen for Pet-1 binding sites in genomic DNA. In, one embodiment cDNA encoding the invention may be used in microchip assays. The present invention contemplates a method of screening, comprising: a) providing in any order: i) a first solid support (e.g. microchip) comprising DNA from a DNA library of the species to be examined and ii) a peptide, or portion thereof, encoded by the DNA of SEQ ID NO:1; b) contacting said microassay microchips with said peptide under conditions such that hybridization can take place.
The present invention may also be used to identify new transcription factors that function in central serotonin 5-HT neurons. In one embodiment, antibodies generated to translation products of the invention may be used in immunoprecipitation experiments to isolate novel transcription factors that interact with Pet-1. In another embodiment, the invention may be used to generate fusion proteins that could also be used to isolate novel transcription factors or other interactive proteins. In yet another embodiment, screens may be conducted using the yeast two-hybrid system.
The present invention may also be used to identify new homologs of Pet-1 or natural mutations thereof. The present invention contemplates screening for homologs using standard molecular procedures. In one embodiment screens are conducted using Northern and Southern blotting.
The present invention contemplates a method of screening a compound, said method comprising: a) providing in any order: i) a first group of cells comprising a recombinant expression vector, wherein said vector comprises at least a portion of the oligonueleotide sequence of SEQ ID NO:1, ii) a second group of cells comprising a recombinant expression vector, wherein said vector comprises a suitable control (i.e. an empty vector), and iii) at least one compound suspected of having the ability to modulate central serotonin 5-HT neuron activity; b) contacting said first and second groups of cells with said compound; and c) detecting the effects of said compound by screening for seronergic receptor generation or serotonin release by methods know to those practiced in the art.
The present invention also contemplates a method of screening for homologs, said method comprising: a) providing in any order: i) a nucleic acid comprising at least a portion of the sequence of SEQ ID NO: 1, and ii) DNA libraries from cells or tissues suspected to comprise said homolog; and b) hybridizing said first or second nucleic acid with said DNA of said library under conditions such that said DNA suspected of coding for said homolog is detected.
The present invention also contemplates a method of screening for interactive peptides, said method comprising: a) providing in any order: i) a peptide comprising at least a portion of the peptide sequence of SEQ ID NO: 2 (including but not limited to portions that are part of fusion proteins, i.e. proteins that contain another portion, such as a portion useful for protein purification) and b) an extract from source (e.g. cells or tissues) suspected of having said interactive peptides; and c) mixing said peptide with said extract under conditions such that said interactive peptide is detected.
The present invention contemplates another approach for screening for interactive peptides, said method comprising: a) providing in any order: i) antibodies reactive with (and usually specific for) at least a portion of a peptide having the sequence of SEQ ID NO: 2, and ii) an extract from a source (e.g. cells or tissues) suspected of having said interactive peptide(s); and b) mixing said antibody with said extract under conditions such that said interactive peptide is detected.
The present invention contemplates the generation of cell lines that express the Pet-1 gene, or portion thereof. The present invention is not limited to any particular cell line. Neuronal cells may be used. Likewise, non-neuronal cells may be used to establish a cell line that Pet-1 expression can be studied without interference of neuronal processes.
The present invention contemplates DNA binding assays where a) Pet-1 DNA (SEQ ID NO:1), or portion thereof, is either i) adhered to a solid support surface or ii) placed in a suspension, b) compounds suspected of binding to the DNA are added in a manner that promotes binding and c) binding is measured by methods known to those practiced in the art. Detection methods utilized may be, but are not limited to, staining, gel electrophoresis and spectrophometric methods.
The present invention contemplates high through put screening methods. Such methods may include, but are not limited to, DNA array assays, spectrophotometric assays, the use of robotics, the use of computerized assay systems and the use of commercially available systems.
The present invention contemplates screening for proteins that bind to Pet-1 binding sites selected from the group comprising SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 (Table 1). The present invention is not limited to any particular assay method. In one embodiment, DNA encoding these sequences is attached to a solid surface (i.e. a microchip) and protein suspected of binding the DNS sequences is placed in contact with the DNA. Attached proteins are then analysed by methods know to those in the art.
To facilitate understanding of the invention, a number of terms are defined below.
As used herein xe2x80x9cagentxe2x80x9d, xe2x80x9ccompoundxe2x80x9d or xe2x80x9cdrugxe2x80x9d is used herein to denote a compound or mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues that are suspected of having therapeutic properties. The compound, agent or drug may be purified, substantially purified or partially purified.
As used herein xe2x80x9cagonistxe2x80x9d refers to molecules or compounds which mimic the action of a xe2x80x9cnativexe2x80x9d or xe2x80x9cnaturalxe2x80x9d compound. Agonists may or may not be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, agonists may or may not be recognized by, e.g., receptors expressed on cell surfaces. In any event, regardless if the agonist is recognized by a natural compound in a manner similar to a xe2x80x9cnaturalxe2x80x9d compound or molecule, the agonist may cause physiologic and/or biochemical changes within the cell, such that the cell reacts to the presence of the agonist in the same manner as if the natural compound was present.
As used herein xe2x80x9cantagonistxe2x80x9d refers to molecules or compounds which inhibit the action of a xe2x80x9cnativexe2x80x9d or xe2x80x9cnaturalxe2x80x9d compound. Antagonists may or may not be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, antagonists may be recognized by the same or different receptors or molecules that are recognized by an agonist. Antagonists may have allosteric effects which prevent the action of an agonist (e.g., by modifying a DNA adduct, or antagonists may prevent the function of the agonist (e.g., by blocking a DNA repair molecule).
As used herein, the term xe2x80x9cpurifiedxe2x80x9d or xe2x80x9cto purifyxe2x80x9d refers to the removal of some contaminants from a sample. The present invention contemplates purified compositions (discussed above).
As used herein, the term xe2x80x9cpartially purifiedxe2x80x9d refers to the removal of a moderate portion of the contaminants of a sample to the extent that the substance of interest is recognizable by techniques known to those skilled in the art as accounting for a measurable amount of the mixture.
As used herein, the term xe2x80x9csubstantially purifiedxe2x80x9d refers to the removal of a significant portion of the contaminants of a sample (e.g.  greater than 90%) to the extent that the substance of interest is recognizable by techniques known to those skilled in the art as the most abundant substance in the mixture.
As used herein the term xe2x80x9cportionxe2x80x9d when in reference to a protein (as in xe2x80x9ca portion of a given proteinxe2x80x9d) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. In one embodiment, the present invention contemplates xe2x80x9cfunctional portionsxe2x80x9d of a protein. Such portions are xe2x80x9cfunctionalxe2x80x9d if they contain a binding region (i.e. a region having affinity for another molecule) and such binding can take place (i.e. the binding region functions, albeit with perhaps lower affinity than that observed for the full-length protein). Such xe2x80x9cfunctional portionsxe2x80x9d of the gene product are typically greater than 10 amino acids in length, and more typically greater than 50 amino acids in length, and even more typically greater than 100 amino acids in length. xe2x80x9cFunctional portionsxe2x80x9d may also be xe2x80x9cconserved portionsxe2x80x9d of the protein. The alignment of the various gene products permit one skilled in the art to select conserved portions of the protein (i.e. those portions in common between two or more species) as well as unconserved portions (i.e. those portions unique to two or more species). The present invention contemplates conserved portions 10 amino acids in length or greater, and more typically greater than 50 amino acids in length.
The present invention contemplates the Pet-1 gene in operable combination with a promoter. xe2x80x9cIn operable combinationxe2x80x9d, xe2x80x9cin operable orderxe2x80x9d and xe2x80x9coperably linkedxe2x80x9d as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
xe2x80x9cPatientxe2x80x9d shall be defined as a human or other animal, such as a guinea pig or mouse and the like, capable of having cell cycle (influenced) determined diseases, either naturally occurring or induced, including but not limited to cancer.
The term xe2x80x9cgenexe2x80x9d refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or its precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence.
The term xe2x80x9cnucleic acid sequence of interestxe2x80x9d refers to any nucleic acid sequence the manipulation of which may be deemed desirable for any reason by one of ordinary skill in the art.
The term xe2x80x9crecombinantxe2x80x9d when made in reference to a DNA molecule refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques. The term xe2x80x9crecombinantxe2x80x9d when made in reference to a protein or a polypeptide refers to a protein molecule which is expressed using a recombinant DNA molecule.
As used herein, the terms xe2x80x9cvectorxe2x80x9d and xe2x80x9cvehiclexe2x80x9d are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
The term xe2x80x9cexpression constructxe2x80x9d, xe2x80x9cexpression vectorxe2x80x9d or xe2x80x9cexpression cassettexe2x80x9d as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
The term xe2x80x9chybridizationxe2x80x9d as used herein refers to any process by which a strand of nucleic acid joins with a complementary strand through base pairing.
As used herein, the terms xe2x80x9ccomplementaryxe2x80x9d or xe2x80x9ccomplementarityxe2x80x9d when used in reference to polynucleotides refer to polynucleotides which are related by the base-pairing rules. For example, for the sequence 5xe2x80x2-AGT-3xe2x80x2 is complementary to the sequence 5xe2x80x2-ACT-3xe2x80x2. Complementarity may be xe2x80x9cpartial,xe2x80x9d in which only some the nucleic acidsxe2x80x2 bases are matched according to the base pairing rules. Or, there may be xe2x80x9ccompletexe2x80x9d or xe2x80x9ctotalxe2x80x9d complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
The term xe2x80x9chomologyxe2x80x9d when used in relation to nucleic acids refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term xe2x80x9csubstantially homologous.xe2x80x9d The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a sequence which is completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
Low stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42xc2x0 C. in a solution consisting of 5X SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt""s reagent [50X Denhardt""s contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 xcexcg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42xc2x0 C. when a probe of about 500 nucleotides in length is employed.
High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42xc2x0 C. in a solution consisting of 5X SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt""s reagent and 100 xcexcg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1X SSPE, 1.0% SDS at 42xc2x0 C. when a probe of about 500 nucleotides in length is employed.
When used in reference to nucleic acid hybridization the art knows well that numerous equivalent conditions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above listed conditions.
xe2x80x9cStringencyxe2x80x9d when used in reference to nucleic acid hybridization typically occurs in a range from about Tmxe2x88x925xc2x0 C. (5xc2x0 C. below the Tm of the probe) to about 20xc2x0 C. to 25xc2x0 C. below Tm. As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. Under xe2x80x9cstringent conditionsxe2x80x9d a nucleic acid sequence of interest will hybridize to its exact complement and closely related sequences.
As used herein, the term xe2x80x9cfusion proteinxe2x80x9d refers to a chimeric protein containing the protein of interest (i.e., Pet-1 and fragments thereof) joined to an exogenous protein fragment (the fusion partner which consists of a non-Pet-1 sequence). The fusion partner may provide a detectable moiety, may provide an affinity tag to allow purification of the recombinant fusion protein from the host cell, or both. If desired, the fusion protein may be removed from the protein of interest by a variety of enzymatic or chemical means known to the art.
xe2x80x9cAntibodyxe2x80x9d shall be defined as a glycoprotein produced by B cells that binds with high specificity to the agent (usually, but not always, a peptide), or a structurally similar agent, that generated its production. Antibodies may be produced by any of the known methodologies (reference) and may be either polyclonal or monoclonal.
The phrase xe2x80x9cgain-of-functionxe2x80x9d (gof) as used herein is applicable to the situation where a modified oligonucleotide that, when transfected into a host organism and translated into a peptide, results in a peptide that will function with increased efficiency (e.g. rate of reaction, affinity, etc.) as compared to the wild type peptide. For example, the modified oligonucleotide (or xe2x80x9cgof nucleotidexe2x80x9d) may, in effect, function as an augmenter of the natural gene if the natural gene is present and functional in the host into which the gof oligonucleotide was transfected, or it may add that function to the host if the natural gene is not present or is non-functional.
The phrase xe2x80x9closs-of-functionxe2x80x9d (lof) as used herein is applicable to the situation where a modified oligonucleotide, when transfected into a host organism and translated into a peptide, results in a peptide that function with decreased efficiency (e.g. rate of reaction, affinity, etc.) as compared to the wild type peptide. For example, the modified oligonucleotide (or xe2x80x9clofxe2x80x9d oligonucleotidexe2x80x9d) may, in effect, function as a diminisher of natural gene function if the natural gene is present and functional in the host into which the lof oligonucleotide was transfected, or may negatively interfere with processes in the host if the natural gene is not present or is non-functional.